Cancer or malignancies can be classified as solid or hematopoietic. Examples of the first ones include cancer such as breast, head and neck, colon and rectum cancer, among others. Examples of the hematologic type include leukemia and lymphomas. The DNA in the cellular nucleus exists arranged in chromatin, and has several levels of order. The constitutive unit of chromatin is the nucleosome that consists of octameric nuclear proteins known as histones and on which the DNA winds. The arranging or packing of DNA in the nucleosomes plays an important role for gene regulation. Covalent modifications of histones such as acetylation, play a fundamental role for chromatin regulation and gene expression (Cho K S, Elizondo L I, Boerkoel C F: Advances in chromatin remodeling and human disease. Curr Opin Genet Dev 2004; 14:308-15).
Currently, cancer remains as a significant health problem world-wide, according to the International Cancer Research Agency and to World Health Organization, the incidence of this disease is increasing dramatically, with an estimation that of 10 million new cases that were observed in the year 2000, in 20 more years there will be 15 millions. (Mignogna M D, Fedele S, Russo L L. The World Cancer Report and the burden of oral cancer. Eur J Cancer Prev. 2004; 13: 139-42). On the other hand, the survival of patients suffering from the most common cancers such as lung, prostate and breast cancer has improved discreetly in the last years. The survival to 5 years was 50% in 1974 and it increased to 63% in the period from 1992 to 1999 (Jemal A, Murray T, Ward E, Samuels A, Tiwari R C, Ghafoor A, Feuer E J, Thun M J. Cancer statistics, 2005. CA Cáncer J. Clin. 2005; 55:10-30).
Although the advances in the forms of treatment have allowed small benefits regarding survival, the results are still far from being optimal. At present, chemotherapy together with surgery and radiotherapy are still the fundamental pillar of treatment since the immense majority of patients with cancer need this form of therapy.
The vast knowledge generated on the molecular basis of cancer in the last years, has permitted the design of new forms of therapy that generally are directed to block the function of oncogenes or to reactivate the expression of suppressive genes. Exemplary of these efforts are the use of monoclonal antibodies against some oncogenic receptors such as EGFR, HER2, etc. In case of the suppressive genes, some therapeutic efforts are the use of recombinant adenovirus harboring the coding gene to the functional product of p53 (Hermiston T W, Kirn D H. Genetically based therapeutics for cancer: similarities and contrasts with traditional drug discovery and development. Mol Ther. 2005; 11:496-507).
Generally speaking, we can call to this new form of cancer as directed to a single gene product or single gene therapy. However, this approach has severe drawbacks because the malignant cell genome is tremendously adaptable and because the nature of cancer is of multiple steps, therefore, there does not exist a single genetic alteration responsible for the development of the malignant phenotype. This mean that, although the blocking or restitution of a gene or its product can produce an important antitumoral effect, said effect is not supported since with all certainty, the malignant cell eventually will develop resistance against said therapy since the malignant cell will increase or decrease the expression of genes than can accommodate the effect caused by said therapy (Ross J S, Schenkein D P, Pietrusko R, Rolfe M, linette G P, Stec J, Stagliano N E, Ginsburg G S, Symmans W F, Pusztai L, Hortobagyi G N. Targeted therapies for cancer 2004. Am J Clin Pathol. 2004; 122:598-609).
At present, it is well known that malignant cells have multiple defects, namely mutations, deletions, duplications, amplifications, as well as epigenetic changes, the latter being stable functional changes due mainly to chromatin modifications, the two more important changes being DNA methylation and histone acetylation. Epigenetic changes must be in a certain state and act in a perfect functional balance to maintain the “malignant homeostasis”. This concept is very important since all the defects of the malignant cells are not simply summations, this is consistent with the fact that the proteins coded by the genes play multiple rolls in networks of complex and interactive functions that shows controls of positive and negative feedback. In addition, through the multiple steps process that occurs in tumor generation, the cell should maintain a steady state between the positive and negative signals both from the oncogenic routes and from suppressive genes, to assure that the processes of proliferation and cellular death occur according to the malignant state dynamics (Weinstein I B. Cancer. Addiction to oncogenes—the Achilles heal of cancer. Science. 2002; 297(5578):63-4).
In addition to the inherent complexity of global genic expression of malignant cells, the picture is much more complicated when trying to regulate the genic expression as a consequence of exogenous stimuli, particularly from the effect of chemotherapy or radiotherapy. Chemotherapy and radiotherapy produce immediate changes in genic transcription, and these changes occur not only in those genes primarily relevant to the carcinogenic process but also in genes that do not take part directly, such as those involved in metabolism, transport, etc. (Alaoui-Jamali M A, Dupre I, Qiang H. Prediction of drug sensitivity and drug resistance in cancer by transcriptional and proteomic profiling. Drug Resist Updat. 2004; 7:245-55). The most important aspect, however, is that only those cells capable of having an adequate transcriptional response to the harmful stimulus are the only ones that will survive the insult. Clearly, in order for this response “adapted” to survive occurs, it is indispensable that the epigenetic mechanisms that regulate transcription are intact. Therefore, if the malignant cell is under the influence of transcriptome-modifying agents, the transcriptional response necessary for survival will not occur and the cell might have functional irreversible changes or suffer apoptosis. The transcription of eukaryotic cells can be defined as the ability of said cells to express biologically active proteins. Therefore, the transcription is a highly regulated phenomenon. The process initiates at gene level and terminates at protein level and involves multiple events; hence the transcription has several levels of regulation among which they are the following: 1) chromatin structure, which is the physical structure of DNA that includes the level of chromatin packing which determines the ability of regulatory proteins to bind to gene regulatory or promoter regions, 2) control of initiation of transcription, 3) transcript transport, 4) transcript processing and modification, 5) transcript stability, 6) translation initiation, 7) post-translational changes, and 8) the transport and stability of the protein. (Archambault J, Friesen J D. Genetics of eukaryotic RNA polymerases I, II, and III. Microbiol. Rev. 1993; 57:703-24). Undoubtedly, the transcriptional effects will be more important if acting higher on the level of transcription regulation. Thus the transcriptome-modifying agents, by acting in the highest level of regulation, will have an effect on the overall genic expression of great magnitude.
Covalent modifications of histones, such as acetylation, and DNA methylation, have an essential role in determining the grade of chromatin packing and finally in determining the overall genic expression; because of that, the agents that inhibit DNA methylation and histone deacetylation have demonstrated to have the property of altering significantly the expression. The loss of methylation might reduce the number of protein complexes that bind to methylated domains in certain locus, leading to a decrease of histone deacetylases activity to which the histone deacetylase-inhibiting agent would have to inhibit. Also, the loss of transcription-repressing complexes can favor the re-association of gene promoters with transcription-activating complexes possessing histone acetylase activity. It is known that the most abundant form of DNA methyl transferase (DNMT1) can bind directly to histone deacetylases and that the amino terminus also has the ability to bind co-repressors {Nakao M. Epigenetics: interaction of DNA methylation and chromatin. Gene. 2001; 278:25-31; Robertson K D. DNA methylation and chromatin-unraveling the tangled web. Oncogene. 2002; 21:5361-79).
Although hundreds of potential antitumoral agents had been tested, the treatment of the human cancer is still challenging, with many of the antitumoral treatments being only partially effective and with the potential of causing collateral effects to practically all the systems. Therefore, there is the need not only to have more effective therapeutic options but also to have some more specific therapies that attack the malignant cells in a more selective way on the genic transcription. For this reason, one of the objects of the present invention is to provide a composition to assist with the treatment of the cancer based on the alteration of the transcriptome by means of the use of transcriptional modifying agents such as hydralazine and magnesium valproate, with which the cells will become unable to survive to harmful stimulus induced by chemotherapy o radiotherapy.
The antihypertensive agent hydralazine is a DNA methylation inhibitor that has been used to hypomethylate T cell DNA in experimental systems which makes these cells auto-reactive (Yung R, Chang S, Hemati N, Johnson K, Richardson B. Mechanisms of drug-induced lupus. IV. Comparison of procainamide and hydralazine with analogs in vitro and in vivo. Arthritis Rheum. 1997; 40:1436-43). More recently, it has been demonstrated that hydralazine produces demethylation of promoter region from suppressive suppressive genes and induces its reactivation in vitro and in vivo models; and also, the genic products reactivated are functional (Segura B, Trejo-Becerril C, Pérez E, Chavez A, Salazar A M, Lizano M, Dueñas-González A. Reactivation of tumor suppressor genes by the cardiovascular drugs hydralazine and procainamide and their potential use in cancer therapy. Clin Cancer Res. 2003; 9: 1596-603).
Hydralazine has direct inhibitory effects on DNA methyl transferase, and in an in vitro model it has been demonstrated that two nitrogen atoms of the molecule interact with the amino acids Lys162 and Arg240 of enzyme active site which accounts for the demethylating and reactivating properties of the function of suppressive genes (Angeles E E, Vazquez-Valadez, V H, Vasquez-Valadez O, Velazguez-Sanchez A M, Ramirez A, Martinez L, Diaz-Barriga S, Romero-Rojas A, Cabrera G, Lopez-Castañares R, Duenas-Gonzalez A: Computational studies of 1-hydrazinophthalazine (Hydralazine) as antineoplasic agent. Docking studies on methyltransferase. Letters Drug Design Discovery 2005; 4:282-286).
Valproic acid or its salts, as in the case of magnesium valproate known also as VPA, 2-propylpentanoic acid is a drug that has been used as anticonvulsivant for many years with a well demonstrated safety profile. (Perucca E: Pharmacological and therapeutic properties of valproate: a summary after 35 years of clinical experience. CNS Drugs 2002, 16:695-714). Recently it has been demonstrated that this medicament is an inhibitor of histone deacetylases. The inhibited enzymes are those of the I and II family class the with exception of the histone desacetylases 6 and 10. Hyperacetylation of histones H3 and H4 observed in vitro and in vivo accompanies its enzymatic inhibitory effect on this family of enzymes. This action on histones produces an important effect on induction of differentiation, induction of apoptosis and inhibition of cellular proliferation (Gurvich N, Tsygankova O M, Meinkoth J L, Klein P S: Histone deacetylase is a target of valproic acid-mediated cellular differentiation. Cancer Res 2004, 64:1079-1086).
For this reason, another treatment kit is disclosed that includes hydralazine and valproic acid or any of its salts such as magnesium valproate, which contributes to the usual therapy used against cancer.